Double strand break points can occur in the genome of a cell through a variety of different mechanisms. For example, chemical mutagens, high intensity magnetic fields, or ionizing radiation can introduce double strand break points. As another example, double strand break points can be introduced into a genome by various genome editing reagents or methods.
An ideal genome editing reagent would only create a double strand break point at one or more “on-target” sites in the genome. However, current genome editing reagents often generate double-stranded break points at one or more “off-target” sites in the genome. In some cases, such off-target sites are highly homologous to an on-target site. For a genome of known sequence and having a small number of highly homologous off-target sites, such highly homologous off-target sites can be identified and assayed for editing using any number of available methods. However, the position, number, and frequency of off-target editing is generally not predictable. In addition, double-stranded breaks introduced by chemical mutagens, high intensity magnetic fields, or ionizing radiation are by their nature unpredictable.
Definitive identification and quantitation of double-stranded breaks throughout the genome of a cell can be performed by whole genome next generation sequencing. However, this can be quite costly and time-consuming, and can be impractical for assaying a population of cells (e.g., a population of cells obtained from a sample of an organism), or for optimizing genome editing reagents and methods.